Georgina Hernández-Chávez

A family of removable cassettes designed to obtain antibiotic-resistance-free genomic modifications of Escherichia coli and other bacteria

Modifications of microbial genomes often require the use of the antibiotic-resistance (Anb(R))-encoding genes and other easily selectable markers. We have developed a set of such selectable markers (Cm(R), Km(R) and Gm(R)), which could easily be inserted into the genome and subsequently removed by using the Cre/loxP site-specific recombination system of bacteriophage P1. In this manner the same marker could be used more than once in the same background, while the resulting strain could or would remain Anb(R) marker-free. Three plasmids were constructed, each containing a cassette consisting of the Cm(R), Km(R), or Gm(R) gene flanked by two parallel loxP sites and two polylinkers (MCS). To test insertion and excision, cassettes were inserted into the lacZ or galE genes carried on an origamma/pir-dependent suicide plasmid, which contained a dominant Sm(R) gene. The cassettes were crossed into the E. coli genome by homologous recombination (allelic exchange), in a manner analogous to that described by Pósfai et al. [Nucl. Acids Res. 22 (1994) 2392-2398], selecting for the Cm(R), Km(R), or Gm(R), for the LacZ(-) or GalE(-) and for the Sm(S) phenotypes (the latter to assure allelic exchange rather than insertion of the entire plasmid). When required, after selecting the strain with the desired modification, the Cm(R), Km(R), or Gm(R) marker was excised by supplying the Cre function. Cre was provided by the thermosensitive plasmid pJW168, which was transformed into the Anb(R) host at 30 degrees C, and was subsequently eliminated at 42 degrees C. Thus the Anb(R) marker was removed, whereas the lacZ or galE gene remained interrupted by the retained loxP site.

New insights into Escherichia coli metabolism: carbon scavenging, acetate metabolism and carbon recycling responses during growth on glycerol

BackgroundGlycerol has enhanced its biotechnological importance since it is a byproduct of biodiesel synthesis. A study of Escherichia coli physiology during growth on glycerol was performed combining transcriptional-proteomic analysis as well as kinetic and stoichiometric evaluations in the strain JM101 and certain derivatives with important inactivated genes.ResultsTranscriptional and proteomic analysis of metabolic central genes of strain JM101 growing on glycerol, revealed important changes not only in the synthesis of MglB, LamB and MalE proteins, but also in the overexpression of carbon scavenging genes: lamB, malE, mglB, mglC, galP and glk and some members of the RpoS regulon (pfkA, pfkB, fbaA, fbaB, pgi, poxB, acs, actP and acnA). Inactivation of rpoS had an important effect on stoichiometric parameters and growth adaptation on glycerol. The observed overexpression of poxB, pta, acs genes, glyoxylate shunt genes (aceA, aceB, glcB and glcC) and actP, suggested a possible carbon flux deviation into the PoxB, Acs and glyoxylate shunt. In this scenario acetate synthesized from pyruvate with PoxB was apparently reutilized via Acs and the glyoxylate shunt enzymes. In agreement, no acetate was detected when growing on glycerol, this strain was also capable of glycerol and acetate coutilization when growing in mineral media and derivatives carrying inactivated poxB or pckA genes, accumulated acetate. Tryptophanase A (TnaA) was synthesized at high levels and indole was produced by this enzyme, in strain JM101 growing on glycerol. Additionally, in the isogenic derivative with the inactivated tnaA gene, no indole was detected and acetate and lactate were accumulated. A high efficiency aromatic compounds production capability was detected in JM101 carrying pJLBaroGfbrtktA, when growing on glycerol, as compared to glucose.ConclusionsThe overexpression of several carbon scavenging, acetate metabolism genes and the absence of acetate accumulation occurred in JM101 cultures growing on glycerol. To explain these results it is proposed that in addition to the glycolytic metabolism, a gluconeogenic carbon recycling process that involves acetate is occurring simultaneously in this strain when growing on glycerol. Carbon flux from glycerol can be efficiently redirected in JM101 strain into the aromatic pathway using appropriate tools.

Metabolic engineering for improving anthranilate synthesis from glucose in Escherichia coli

BackgroundAnthranilate is an aromatic amine used industrially as an intermediate for the synthesis of dyes, perfumes, pharmaceuticals and other classes of products. Chemical synthesis of anthranilate is an unsustainable process since it implies the use of nonrenewable benzene and the generation of toxic by-products. In Escherichia coli anthranilate is synthesized from chorismate by anthranilate synthase (TrpED) and then converted to phosphoribosyl anthranilate by anthranilate phosphoribosyl transferase to continue the tryptophan biosynthetic pathway. With the purpose of generating a microbial strain for anthranilate production from glucose, E. coli W3110 trpD9923, a mutant in the trpD gene that displays low anthranilate producing capacity, was characterized and modified using metabolic engineering strategies.ResultsSequencing of the trpED genes from E. coli W3110 trpD9923 revealed a nonsense mutation in the trpD gene, causing the loss of anthranilate phosphoribosyl transferase activity, but maintaining anthranilate synthase activity, thus causing anthranilate accumulation. The effects of expressing genes encoding a feedback inhibition resistant version of the enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (aroGfbr), transketolase (tktA), glucokinase (glk) and galactose permease (galP), as well as phosphoenolpyruvate:sugar phosphotransferase system (PTS) inactivation on anthranilate production capacity, were evaluated. In shake flask experiments with minimal medium, strains W3110 trpD9923 PTS- and W3110 trpD9923/pJLBaroGfbrtkt A displayed the best production parameters, accumulating 0.70–0.75 g/L of anthranilate, with glucose-yields corresponding to 28–46% of the theoretical maximum. To study the effects of extending the growth phase on anthranilate production a fed-batch fermentation process was developed using complex medium, where strain W3110 trpD9923/ pJLBaroGfbrtkt A produced 14 g/L of anthranilate in 34 hours.ConclusionThis work constitutes the first example of a microbial system for the environmentally-compatible synthesis of anthranilate generated by metabolic engineering. The results presented here, including the characterization of mutation in the trpD gene from strain W3110 trpD9923 and the development of a fermentation strategy, establish a step forward towards the future improvement of a sustainable process for anthranilate production. In addition, the present work provides very useful data regarding the positive and negative consequences of the evaluated metabolic engineering strategies.

Metabolic Engineering of Escherichia coli for l-Tyrosine Production by Expression of Genes Coding for the Chorismate Mutase Domain of the Native Chorismate Mutase-Prephenate Dehydratase and a Cyclohexadienyl Dehydrogenase from Zymomonas mobilis

ABSTRACT The expression of the feedback inhibition-insensitive enzyme cyclohexadienyl dehydrogenase (TyrC) from Zymomonas mobilis and the chorismate mutase domain from native chorismate mutase-prephenate dehydratase (PheACM) from Escherichia coli was compared to the expression of native feedback inhibition-sensitive chorismate mutase-prephenate dehydrogenase (CM-TyrAp) with regard to the capacity to produce l-tyrosine in E. coli strains modified to increase the carbon flow to chorismate. Shake flask experiments showed that TyrC increased the yield of l-tyrosine from glucose (Yl-Tyr/Glc) by 6.8-fold compared to the yield obtained with CM-TyrAp. In bioreactor experiments, a strain expressing both TyrC and PheACM produced 3 g/liter of l-tyrosine with a Yl-Tyr/Glc of 66 mg/g. These values are 46 and 48% higher than the values for a strain expressing only TyrC. The results show that the feedback inhibition-insensitive enzymes can be employed for strain development as part of a metabolic engineering strategy for l-tyrosine production.

Production of cinnamic and p -hydroxycinnamic acid from sugar mixtures with engineered Escherichia coli

BackgroundThe aromatic compounds cinnamic acid (CA) and p-hydroxycinnamic acid (pHCA) are used as flavoring agents as well as precursors of chemicals. These compounds are present in plants at low concentrations, therefore, complex purification processes are usually required to extract the product. An alternative production method for these aromatic acids is based on the use of microbial strains modified by metabolic engineering. These biotechnological processes are usually based on the use of simple sugars like glucose as a raw material. However, sustainable production processes should preferably be based on the use of waste material such as lignocellulosic hydrolysates.ResultsIn this study, E. coli strains with active (W3110) and inactive phosphoenolpyruvate:sugar phosphotransferase system (PTS) (VH33) were engineered for CA and pHCA production by transforming them with plasmids expressing genes encoding phenylalanine/tyrosine ammonia lyase (PAL/TAL) enzymes from Rhodotorula glutinis or Arabidopsis thaliana as well as genes aroGfbr and tktA, encoding a feedback inhibition resistant version of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and transketolase, respectively. The generated strains were evaluated in cultures with glucose, xylose or arabinose, as well as a simulated lignocellulosic hydrolysate containing a mixture of these three sugars plus acetate. Production of CA was detected in strains expressing PAL/TAL from A. thaliana, whereas both CA and pHCA accumulated in strains expressing the enzyme from R. glutinis. These experiments identified arabinose and W3110 expressing PAL/TAL from A. thaliana, aroGfbr and tktA as the carbon source/strain combination resulting in the best CA specific productivity and titer. To improve pHCA production, a mutant with inactive pheA gene was generated, causing an 8-fold increase in the yield of this aromatic acid from the sugars in a simulated hydrolysate.ConclusionsIn this study the quantitative contribution of active or inactive PTS as well as expression of PAL/TAL from R. glutinis or A. thaliana were determined for production performance of CA and pHCA when growing on carbon sources derived from lignocellulosic hydrolysates. These data will be a useful resource in efforts towards the development of sustainable technologies for the production of aromatic acids.

Metabolic regulation analysis of an ethanologenic Escherichia coli strain based on RT-PCR and enzymatic activities

BackgroundA metabolic regulation study was performed, based upon measurements of enzymatic activities, fermentation performance, and RT-PCR analysis of pathways related to central carbon metabolism, in an ethanologenic Escherichia coli strain (CCE14) derived from lineage C. In comparison with previous engineered strains, this E coli derivative has a higher ethanol production rate in mineral medium, as a result of the elevated heterologous expression of the chromosomally integrated genes encoding PDCZmand ADHZm(pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis). It is suggested that this behavior might be due to lineage differences between E. coli W and C.ResultsThis study demonstrated that the glycolytic flux is controlled, in this case, by reactions outside glycolysis, i.e., the fermentative pathways. Changes in ethanol production rate in this ethanologenic strain result in low organic acid production rates, and high glycolytic and ethanologenic fluxes, that correlate with enhanced transcription and enzymatic activity levels of PDCZmand ADHZm. Furthermore, a higher ethanol yield (90% of the theoretical) in glucose-mineral media was obtained with CCE14 in comparison with previous engineered E. coli strains, such as KO11, that produces a 70% yield under the same conditions.ConclusionResults suggest that a higher ethanol formation rate, caused by ahigher PDCZmand ADHZmactivities induces a metabolic state that cells compensate through enhanced glucose transport, ATP synthesis, and NAD-NADH+H turnover rates. These results show that glycolytic enzymatic activities, present in E. coli W and C under fermentative conditions, are sufficient to contend with increases in glucose consumption and product formation rates.

Specific ethanol production rate in ethanologenic Escherichia coli strain KO11 Is limited by pyruvate decarboxylase.

Modification of ethanol productivity and yield, using mineral medium supplemented with glucose or xylose as carbon sources, was studied in ethanologenic Escherichia coli KO11 by increasing the activity of five key carbon metabolism enzymes. KO11 efficiently converted glucose or xylose to ethanol with a yield close to 100% of the theoretical maximum when growing in rich medium. However, when KO11 ferments glucose or xylose in mineral medium, the ethanol yields decreased to only 70 and 60%, respectively. An increase in GALPEc (permease of galactose-glucose-xylose) or PGKEc (phosphoglycerate kinase) activities did not change xylose or glucose and ethanol flux. However, when PDCZm (pyruvate decarboxylase from Zymomonas mobilis) activity was increased 7-fold, the yields of ethanol from glucose or xylose were increased to 85 and 75%, respectively, and organic acid formation rates were reduced. Furthermore, as a response to a reduction in acetate and ATP yield, and a limited PDCZm activity, an increase in PFKEc (phosphofructokinase) or PYKBs (pyruvate kinase from Bacillus stearothermophilus) activity drastically reduced glucose or xylose consumption and ethanol formation flux. This experimental metabolic control analysis showed that ethanol flux in KO11 is negatively controlled by phosphofructokinase and pyruvate kinase, and positively influenced by the PDCZm activity level.

Inactivation of Pyruvate Kinase or the Phosphoenolpyruvate: Sugar Phosphotransferase System Increases Shikimic and Dehydroshikimic Acid Yields from Glucose in Bacillus subtilis

The glycolytic intermediate phosphoenolpyruvate (PEP) is a precursor of several cellular components, including various aromatic compounds. Modifications to the PEP node such as PEP:sugar phosphotransferase system (PTS) or pyruvate kinase inactivation have been shown to have a positive effect on aromatics production capacity in Escherichia coli and Bacillus subtilis. In this study, pyruvate kinase and PTS-deficient B. subtilis strains were employed for the construction of derivatives lacking shikimate kinase activity that accumulate two industrially valuable chemicals, the intermediates of the common aromatic pathway, shikimic and dehydroshikimic acids. The pyruvate kinase-deficient strain (CLC6-PYKA) showed the best production parameters under resting-cell conditions. Compared to the PTS-deficient strain, the shikimic and dehydroshikimic acids specific production rates for CLC6-PYKA were 1.8- and 1.7-fold higher, respectively. A batch fermentor culture using complex media supplemented with 83 g/l of glucose was developed with strain CLC6-PYKA, where final titers of 4.67 g/l (shikimic acid) and 6.2 g/l (dehydroshikimic acid) were produced after 42 h.

Physiological and transcriptional characterization of Escherichia coli strains lacking interconversion of phosphoenolpyruvate and pyruvate when glucose and acetate are coutilized

Phosphoenolpyruvate (PEP) is a precursor involved in the biosynthesis of aromatics and other valuable compounds in Escherichia coli. The PEP:carbohydrate phosphotransferase system (PTS) is the major glucose transport system and the largest PEP consumer. To increase intracellular PEP availability for aromatics production purposes, mutant strains of E. coli JM101 devoid of the ptsHIcrr operon (PB11 strain) have been previously generated. In this derivative, transport and growth rate on glucose decreased significantly. A laboratory evolved strain derived from PB11 that partially recovered its growth capacity on glucose was named PB12. In the present study, we blocked carbon skeletons interchange between PEP and pyruvate (PYR) in these ptsHIcrr− strains by deleting the pykA, pykF, and ppsA genes. The PB11 pykAF− ppsA− strain exhibited no growth on glucose or acetate alone, but it was viable when both substrates were consumed simultaneously. In contrast, the PB12 pykAF− ppsA− strain displayed a low growth rate on glucose or acetate alone, but in the mixture, growth was significantly improved. RT‐qPCR expression analysis of PB11 pykAF− ppsA− growing with both carbon sources showed a downregulation of all central metabolic pathways compared with its parental PB11 strain. Under the same conditions, transcription of most of the genes in PB12 pykAF− ppsA− did not change, and few like aceBAK, sfcA, and poxB were overexpressed compared with PB12. We explored the aromatics production capabilities of both ptsHIcrr− pykAF− ppsA− strains and the engineered PB12 pykAF− ppsA− tyrR− pheAev2+/pJLBaroGfbrtktA enhanced the yield of aromatic compounds when coutilizing glucose and acetate compared with the control strain PB12 tyrR− pheAev2+/pJLBaroGfbrtktA. Biotechnol. Bioeng. 2014;111: 1150–1160.

Physiologic consequences of glucose transport and phosphoenolpyruvate node modifications in Bacillus subtilis 168.

The phosphoenolpyruvate (PEP) node is an important carbon distribution point in the central metabolic networks; therefore, its modification is a common strategy employed for developing microbial production strains. In this study, mutants of Bacillus subtilis 168 were generated with deletions of pykA (which encodes pyruvate kinase), ptsG (which encodes the glucose-specific IICBAGlc component) or the ptsGHI operon [which encodes IICBAGlc, HPr protein and enzyme I from the PEP:sugar phosphotransferase system (PTS)]. These modifications caused a reduction in the initial rate of [14C]-glucose import, corresponding to 10.99, 2.83 and 0.50% of that found in B. subtilis 168 for strains with inactive pykA, ptsG or ptsGHI genes, respectively. Characterization of derivative strains lacking 3-dehydroquinate synthase activity showed that inactivation of pykA leads to an 8-fold increase in carbon flow to the common aromatic pathway. Quantitative real-time PCR analyses of 76 genes from several functional classes revealed a carbon starvation transcriptional pattern that includes a partial gluconeogenic response and overexpression of genes encoding non-PTS glucose importers in the strains lacking functional pykA, ptsG or ptsGHI genes. A transcriptional response consistent with pyruvate limitation was also detected, which includes upregulation of genes encoding malic enzymes that generate pyruvate from malate.